https://orcid.org/0000-0002-4264-1864
Figures
Abstract
Giardia duodenalis is an important extracellular protozoan parasite of the gut responsible for waterborne diarrhea in human and nonhuman animals worldwide. Giardia trophozoites express and secrete excretory-secretory proteins (ESPs) affecting structural, cellular, and soluble components of the host small intestinal milieu, of which tenascins are present in high abundance. Giardia-induced intestinal epithelial cell (IEC) apoptosis is known as an important aspect of pathogenesis of giardiasis, while the underlying molecular mechanisms remain largely unclear. Here we used in vitro models of IECs to explore the regulatory mechanism of Giardia-induced apoptosis. We initially confirmed the occurrence of apoptosis and the activation of epidermal growth factor receptor (EGFR) when IECs were exposed to Giardia trophozoites, and EGFR activation involved Giardia-induced IEC apoptosis. The recombinant Tenascin15, Tenascin30, and Tenascin33 were then studied for their potential to activate EGFR/signal transducer and activator of transcription 3 (STAT3)-dependent IEC apoptosis. All the three recombinant proteins were demonstrated to be effective in triggering IEC apoptosis and EGFR/STAT3 activation. Strikingly, IEC apoptosis induced by rTenascin15 and rTenascin30 were found to be dependent on the activation and nuclear translocation of EGFR and STAT3, while this is not the case for rTenascin33. Collectively, our study identified tenascins as potential virulence factors related to Giardia-induced IEC apoptosis, and demonstrated that EGFR-STAT3 axis played a critical regulatory role in the process, advancing our understanding of the pathogenesis of Giardia infection.
Author summary
IEC apoptosis is a crucial part of giardiasis development and the causative mechanisms behind the process remain poorly understood to date. During Giardia infection, trophozoites adhere tightly to the small intestine, and the physical attachment and the secreted ESPs at the interface of host and parasite interactions are believed to increase the risk of pathogenicity of giardiasis. In this study, Giardia-secreted tenascins rich in EGF-like domains were determined as critical contributors to IEC apoptosis. EGFR-STAT3 axis was then confirmed to be a vital player in tenascins-associated IEC apoptosis. With the findings of this study, the mechanism underlying Giardia-host interactions is becoming increasingly clear. Because EGFR is a commonly used drug target in the clinical practice, its role as a future therapeutic target against Giardia infection deserves in-depth exploration.
Citation: Yu S, Sun Z, Wang Y, Zhang P, Sun H, Meng Z, et al. (2026) The differential roles of Giardia duodenalis-secreted tenascins in inducing intestinal epithelial cell apoptosis and the attributed EGFR-STAT3 axis regulation. PLoS Negl Trop Dis 20(3): e0014153. https://doi.org/10.1371/journal.pntd.0014153
Editor: Maria Fantinatti, Universidade do Estado do Rio de Janeiro, BRAZIL
Received: September 17, 2025; Accepted: March 17, 2026; Published: March 23, 2026
Copyright: © 2026 Yu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: This research was funded by the Natural Science Fund of Heilongjiang Province for Distinguished Young Scholars (JQ2024C001) to Wei Li, the Key Research and Development Program of Heilongjiang Province (2024ZXDXB41) to Wei Li, and the National Natural Science Foundation of China (32172885) to Wei Li. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Giardia duodenalis, also known as G. intestinalis and G. lamblia, is a flagellated protozoan parasite infecting a wide spectrum of vertebrate hosts and causing giardiasis [1]. There exist eight genetic assemblages within G. duodenalis, among which assemblages A and B are considered zoonotic and thereby are of public health relevance [2]. The life of Giardia can be divided into two main stages: the disease-causing vegetative trophozoite and the environmentally resistant and infective cyst [2]. Giardia trophozoites are heart or pear-shaped, with flagella involving motility and sucking discs involving adhesion to host intestinal epithelial cells (IECs) in the small intestine [3]. Giardia cysts are extremely transmissible and ingestion of a minimum of 10 cysts can lead to infection [2]. Mostly, Giardia infections are clinically asymptomatic, the typical clinical symptoms are reported as diarrhea, malaise, flatulence, greasy stools, and abdominal cramps [4]. In some cases, giardiasis can become a chronic disease associated with food allergies, irritable bowel syndrome, chronic fatigue syndrome or arthritis [5–7]. Our current understanding of the pathogenesis of giardiasis is quite limited, the known pathogenic factors include physical attachment at IECs, secretion of virulence factors, immune evasion, and competitive consumption of nutrients [8–11]. Giardia secretes excretory-secretory proteins (ESPs) during its interaction with IECs, some of which function as virulence factors in association with IEC apoptosis, tight junction disruption, and epithelial barrier damage [10,12].
Among the ESPs identified through the proteomics, tenascins are a highly representative and variable group of large extracellular matrix glycoproteins abundantly expressed in the supernatants of cultures of G. duodenalis WB isolate (assemblage A) and GS isolate (assemblage B) [13–16], their role in the pathogenesis of giardiasis remains to be explored. Giardia tenascins are rich in epidermal growth factor (EGF)-like domains [17]. The EGF-like domain contains 30–40 amino acid residues characterized by the arrangement C(cysteine)X(any amino acid)4–14CX3–8CX4–14CXCX8-14C, which represents one of the structural bases for binding to epidermal growth factor receptor (EGFR) [18–20]. High levels of EGF-like domains have been identified in the extracellular milieu, which involve cell signaling and disease phenotypes via protein-protein interactions, and some of the interactions that they mediate constitute potential therapeutic targets [20]. A number of EGF-like domains have structural functions implicated in protein physical properties or ligand attachment. Although many proteins have EGF-like structural domains, only some of those present EGF activity [21]. Those with EGF activity were often identified with limited cysteine spacer in EGF-like domains, as exampled by a human-derived biologically active EGF-like arrangement CX7–8CX4–8CX10–13CXCX8C [18,22,23]. There exist 20 formally described tenascins derived from various isolates of G. duodenalis, among which 11 are confirmed to be secreted [13]. In the present study, three randomly selected WB isolate-derived tenascins with GenBank accession no. GL50803_114815 (63 kDa), GL50803_10330 (28.5 kDa), and GL50803_16833 (66.5 kDa) were probed for their homology with human EGF at the primary structure level, as well as their capability of activating EGFR in human IEC model [13].
EGFR, one of the most well-studied receptor tyrosine kinases, plays a critical role in signaling pathways that regulate a broad variety of cellular functions, such as cell proliferation, differentiation, invasion, and wound healing [24]. Signal transducer and activator of transcription 3 (STAT3) has been found to be of particular importance across a range of scientific fields including infectious diseases, autoimmunity, vaccine response, metabolism, and malignancy [25]. STAT3, together with its family coordinate cytokine and growth factor signaling, are involved in the regulation of a variety of cellular processes like cell proliferation, metabolism, infection, inflammation, and cancer [26]. Apoptosis is vital in the pathogenesis of giardiasis [11,27–34], whereas it remains largely unknown about the regulatory network. EGFR and STAT3 have been intensively investigated for their growth-promoting functions in the context of normal and malignant cellular biology by forming a positive feedback loop [24,25]. Paradoxically, it has been acknowledged that, in some circumstances, STAT3 also operates as a positive mediator of EGFR-dependent apoptosis [35]. Yet, the potential of EGFR-STAT3 axis as a regulator in Giardia-induced IEC apoptosis remains to be elucidated.
Giardia ESPs have been studied for their possible functions during noninvasive infections widely and effectively using in vitro models of IECs, such as enterocytes-like colon cancer cell lines Caco-2 and HT-29 [14]. Upregulation of transcript [36,37] and protein [15] levels of Giardia tenascins has been validated relying on in vitro interactions between Giardia WB isolate and IECs. As mentioned here earlier, there exist potential structural basis for the interaction of Giardia tenascins with EGFR. This study aims to decipher the regulatory function of EGFR-STAT3 axis in Giardia-evoked IEC apoptosis, as well as to explore the possibility for tenascins as a trigger for this cellular process.
Materials and methods
Cell culture
The HT-29 and Caco-2 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in a humidified atmosphere of 5% CO2 at 37°C. HT-29 cells were cultured in DMEM/F12 (Hyclone, Logan, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. Caco-2 cells were grown in high-glucose DMEM (Hyclone, Logan, USA) supplemented with 10% FBS, 1% MEM NEAA, 1% GlutaMAX and 1% penicillin/streptomycin. Cells were passaged every 3–4 days at 80–90% confluence and culture medium was changed every other day. Cells were seeded in 6-well (1 × 106 cells/well), 12-well (5 × 105 cells/well), 24-well (2 × 105 cells/well), and 96-well (1 × 104 cells/well) plates depending on the needs of the experiments. All experiments were performed 2–3 days post-seeding at 80–90% confluence.
Parasite culture and exposure
The trophozoites of G. duodenalis WB isolate (assemblage A; ATCC 30957, Manassas, USA) were axenically cultivated at 37 °C in modified TYI-S-33 culture medium containing 10% FBS and 0.1% bovine bile supplemented with 0.1% gentamycin and 1% penicillin/streptomycin [38]. Unattached parasites were removed by changing the medium. Attached parasites were harvested by chilling on ice for 15 min. Detached trophozoites were centrifuged, washed with PBS, resuspended in culture medium, counted by a hematocytometer, and exposed to IECs at a ratio of 10 parasites/cell. In time-course experiments, parasites were added at different time points and cells harvested together. Prior to further analysis, cells were washed with ice-cold PBS to remove parasites.
Western blot analysis
HT-29 and Caco-2 cells plated in 6-well plates were lysed using RIPA Lysis Solution [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS; Beyotime, Shanghai, China] containing 1% PMSF (Beyotime, Shanghai, China). Protein concentration was determined using a BCA protein assay kit (Beyotime, Shanghai, China). Protein levels were assessed by western blot analysis. In summary, proteins were separated by 12% SDS-PAGE and electro-transferred onto PVDF membranes. Membranes were blocked with 5% skim milk in PBST [137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, and 0.1% (w/v) Tween-20] for 2 h at room temperature (RT) and then incubated overnight at 4 °C with primary antibodies against pro-/cl-CASP3 (1:1000 dilution in PBST), pro-/cl-PARP (1:500), β-actin (1:1000), EGFR (1:800), p-EGFR (1:500), STAT3 (1:500), p-STAT3 (1:500), and LaminB (1:500). The primary antibodies came from two commercial sources (ABclonal, Wuhan, China; Bioss, Beijing, China). Membranes were washed thrice in PBST and probed with HRP-conjugated secondary antibody (1:5000; ABMART, Shanghai, China) for 1 h at RT. Western blot signal was detected using the enhanced chemiluminescent reaction (Thermo Fisher Scientific, Waltham, USA). Protein bands were scanned by the GeneGnome XRQ chemiluminescence imaging system (Syngene, Cambridge, UK; S1 File) and the band intensity was quantified using Image J software (NIH, Bethesda, USA). To assess nuclear translocation of EGFR and STAT3, the cytoplasmic and nuclear fractions were separated and isolated using an NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific, Waltham, USA). The subcellular fractions were then assayed by western blotting.
Acridine orange (AO)/ethidium bromide (EB) assay
HT-29 and Caco-2 cells grown on cover slides in 24-well plates were measured for the occurrence of apoptosis by dual staining with fluorescent dyes AO and EB (BestBio, Shanghai, China) according to the manusfacturer’s instructions and by a Lionheart FX Automated Microscope (BioTek, Winooski, USA). Viable and early apoptotic cells were uniformly stained green, late apoptotic and necrotic cells were stained orange-red. NIH Image J software was used to quantify the fluorescence intensities.
Annexin V-FITC/propidium iodide (PI) apoptosis staining
HT-29 and Caco-2 cells cultured in 12-well plates were harvested and stained with Annexin V-FITC and PI using an Annexin V-FITC/PI Apoptosis Detection Kit (Thermo Fisher Scientific, Waltham, USA). Annexin V has a high affinity for phosphatidylserine present in the plasma membranes of early or late apoptotic cells, and PI identifies late apoptotic and necrotic cells. Briefly, the harvested cells were double-stained with Annexin V-FITC and PI in the dark at RT for 15 min. Samples were analyzed on a BD FACS Canto II flow cytometer equipped with FACSDiva (BD Biosciences, San Jose, USA). Flow cytometry data obtained were analyzed using Flowjo (Tree Star, Ashland, USA).
Cell viability analysis
An additional incubation of cells in FBS-free medium in 96-well plates for 3 h was needed before protein or drug exposure. Cell viability was detected by a cell counting kit-8 (CCK-8; Apexbio, Houston, USA) assay. CCK-8 relies on a water-soluble tetrazolium salt known as WST-8, which can be reduced by cellular dehydrogenases in viable cells to produce a highly water-soluble orange formazan dye. CCK-8 allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays. The optical density was measured at 450 nm.
Protein inhibition
We used EGFR inhibitor Ag1478 (20 μM in use) and STAT3 inhibitor Stattic (10 μM in use) (Selleckchem, Houston, USA) in inhibition analyses (S2 File). Inhibitors were dissolved in 0.1% DMSO, applied 1 h, and washed thrice for drug removal before exposure.
Preparation of recombinant tenascins and exposure
Giardia tenascins are secreted in a free and non-vesicular form and highly expressed in the culture supernatant [13–16]. In this study, Tenascin15 (GL50803_114815), Tenascin30 (GL50803_10330), and Tenascin33 (GL50803_16833) with EGF-like domains derived from G. duodenalis WB isolate (assemblage A) were examined for their ability to activate IEC apoptosis via EGFR-STAT3 axis. The three tenascin proteins have been cloned and expressed using a prokaryotic expression system as described [39]. In brief, the amplified genes encoding the three tenascins mentioned earlier were cloned into pCold I vector (TaKaRa, Ohtsu, Japan). The resulting plasmids were transformed into Escherichia coli strain BL21 (DE3) cells. The recombinant cells were cultured in 250 mL of LB medium supplemented with ampicillin (50 mg/ml) at 37 °C and 200 rpm in a shaking incubator. Protein expression was induced with 1 mM isopropyl β-d-thiogalactoside (Solarbio, Beijing, China) for 20 h at 16 °C [40]. Following induction, bacteria were harvested and lysed. The total proteins after ultrasonication and the supernatants (soluble fractions) after purification using a HisTrap HP nickel column (SMART, Changzhou, China) were subjected to SDS-PAGE analyses (S3 File, panel A). Before exposure, the purified and concentrated recombinant His6-tagged tenascins (S3 File, panel B) were quantified by BCA assay and the protein solution was tested to be free of endotoxins using an endotoxin ELISA kit (Meimian Biotech, Yancheng, China) (S3 File, panel C). In time-course experiments, rTenascin15/30/33 was added at different time points and cells harvested together. Prior to further analysis, non-interacted proteins were removed by washing with PBS.
Co-immunoprecipitation (Co-IP) assay
Protein-protein interactions were examined by Co-IP analysis. In brief, the anti-EGFR antibody (1:100) was initially incubated with protein magnetic A + G beads (Bimake, Houston, USA) at 4 °C overnight with gentle rotation. The immunoprecipitated beads were incubated with lysed cell extracts of HT-29 and then His-tagged tenascins (100 μg/mL) at 4 °C overnight. The immunoprecipitates were washed with lysis buffer and analyzed by western blot analysis with anti-His antibody (1:1000; ABclonal, Wuhan, China) and HRP-conjugated goat anti-mouse secondary antibody (1:5000).
Transmission electron microscopy (TEM) analysis
HT-29 and Caco-2 cells with or without Giardia trophozoite and recombinant tenascin exposure were collected by mild trypsinization and fixed with 2.5% glutaraldehyde overnight at RT. The fixed cells were then embedded in Epon 812 and polymerized at 60 °C for 1 h. Ultrathin sections were cut on an RMC PowerTome XL ultramicrotome at 70 nm (Tucson, USA), stained with 5% uranyl acetate and 2% lead citrate, and examined on a Hitachi H-7650 electron microscope (Tokyo, Japan).
Immunofluorescence assay (IFA)
HT-29 cells grown on cover slips in 24-well plates were fixed with 4% paraformaldehyde in PBS for 30 min at RT and permeabilized with 0.25% Triton X-100 in PBS for 10 min at RT. Nonspecific binding sites were blocked by incubation in 2% BSA in PBS for 30 min at RT. Cells were incubated with anti-His antibody (1:100) at 4 °C overnight and then Alexa Fluor 647-conjugated AffiniPure goat anti-mouse IgG (H + L) (1:200; Bioss, Beijing, China) at 37 °C for 1 h in the dark, or anti-EGFR antibody (1:100) and then FITC-conjugated AffiniPure goat anti-rabbit IgG (H + L) (1:200; Jackson, West Grove, USA) likewise. Cell nuclei were labeled by DAPI (2 μg/mL; Alphabio, Tianjin, China). Fluorescent image was taken by a Lionheart FX Automated Microscope.
Statistical analysis
Statistical analyses were conducted using the GraphPad Prism 7.0 program. Data from triplicate wells from a representative of three independent experiments are presented as means ± SD. The statistical significance of the differences was assessed using Student’s t-test and one-way ANOVA. P-values < 0.05 were considered statistically significant (* p < 0.05, ** p < 0.01).
Results
The involvement of EGFR activation in Giardia-induced IEC apoptosis
Giardia trophozoite exposure induced HT-29 and Caco-2 cell apoptosis as indicated by the elevated levels of apoptotic markers cleaved-PARP and cleaved-CASP3 in a time-dependent manner (Fig 1A), the increasing numbers of apoptotic cells stained orange-red by AO/EB staining (Fig 1B), and a simultaneous increase in the percentage of early and late apoptotic cells by Annexin V-FITC/PI staining (Fig 1C). Increasing EGFR expression of HT-29 and Caco-2 cells was observed following Giardia trophozoite exposure and it peaked at 6 h (Fig 1D and 1E), while increasing EGFR phosphorylation peaked at 30–45 min post exposure (Fig 1D). HT-29 cell apoptosis induced by a 6-h trophozoite exposure could be blunted by blocking EGFR activation using its inhibitor Ag1478 as observed in western blot analysis (Fig 1F), AO/EB analysis (Fig 1G), and Annexin V-FITC/PI analysis (Fig 1H). Those implied involvement of EGFR activation in Giardia-induced IEC apoptosis.
Unless otherwise specified, HT-29 and Caco-2 cells were exposed to Giardia trophozoites for the indicated time periods. (A) Giardia exposure promoted activation of PARP and CASP3 in HT-29 and Caco-2 cells as determined by western blot and gray value analyses. The comparisons were made with the first group of the graph. (B, C) Apoptotic effects on HT-29 and Caco-2 cells by Giardia exposure were assessed by AO/EB staining (scale bar = 1000 μm) and Annexin V-FITC/PI staining. (D, E) Giardia exposure promoted activation of EGFR in HT-29 and Caco-2 cells as determined by western blot and gray value analyses. The comparisons were made with the first group of the graph. (F) PARP and CASP3 activation in HT-29 cells induced by a 6-h Giardia exposure could be reversed by application of the EGFR inhibitor Ag1478 as assessed by western blot and gray value analyses. The comparisons were made as indicated. (G, H) HT-29 cell apoptosis induced by a 6-h Giardia exposure could be reversed by application of the EGFR inhibitor Ag1478 as assessed by AO/EB staining (scale bar = 1000 μm) and Annexin V-FITC/PI staining. Data from triplicate wells from a representative of three independent experiments are presented as means ± SD. * p < 0.05, ** p < 0.01. “Ctr” read “Control”, “GI” read “Giardia”.
EGF-like domain analysis of Giardia-secreted tenascins
We mapped the schematic diagram of EGF-like domains in the primary structures of Tenascin15, Tenascin30 and Tenascin33, and the EGF-like domains in each protein were compared with the sequence from the active domain of a human reference EGF (GenBank accession no. KI723_040922) (Fig 2A). All the three tenascins were shown to contain relatively dense EGF-like domains, with 10 found in Tenascin15, 5 in Tenascin30, and 8 in Tenascin33. Of those in Tenascin33, one from amino acids 223–262 might have EGF activity, since it is strictly in conformity with the human-derived biologically active domain CX7–8CX4–8CX10–13CXCX8C mentioned earlier (Fig 2A). This domain is a well-characterized canonical EGFR ligand widely used as a reference in comparative structural and functional studies as documented [18,22,23]. The effects of rTenascin15/30/33 on the HT-29 cell viability were then measured by CCK-8 assay. HT-29 cell viability was reduced by increasing time of exposure with recombinant proteins from 3 h to 9 h and by increasing concentration of stimuli from 5 μg/mL to 100 μg/mL (Fig 2B–2D). A feasible concentration of 20 μg/mL was then selected and applied for rTenascin15/30/33 exposure in the following investigations. The possible interaction between rTenascin15/30/33 and EGFR was assessed by Co-IP analysis (S4 File).
(A) Schematic diagram exhibiting the EGF-like domains of Tenascin15/30/33 (GenBank accession no. GL50803_114815/10330/16833) derived from G. duodenalis WB isolate (assemblage A) and the alignment of them with a human-derived biologically active EGF-like domain (GenBank accession no. KI723_040922) by ClustalW included in BioEdit v7.0.9 using default parameters [65] and then manual correction. (B, C, D) Effects of rTenascin15/30/33 on cell viability of HT-29 by CCK-8 assay.
rTenascin15/30/33 exposure triggered IEC apoptosis
Considering the enrichment of EGF-like domains in tenascins, we therefore assessed the potential for rTenascin15/30/33 to activate IEC apoptosis. We first used TEM to evaluate apoptotic morphology of HT-29 and Caco-2 cells exposed to Giardia trophozoites and rTenascin15/30/33 for 6 h (Fig 3). Compared to unexposed controls, the exposed HT-29 and Caco-2 cells both exhibited extensive ultrastructural changes characteristic of apoptosis, with massive cytoplasmic vacuolization (indicated by red arrows) observed in both early and late apoptotic cells and apparent nuclear condensation and fragmentation (indicated by yellow arrows) observed in late apoptotic cells (Fig 3). We then evaluated the apoptosis-exacerbating effect of rTenascin15/30/33 at the molecular level. A more significant cleavage of CASP3 and PARP in western blot analysis and a more obvious apoptosis-inducing effect in AO/EB and Annexin V-FITC/PI analyses occurred in HT-29 and Caco-2 cells at 6 h after rTenascin15/30 exposure in comparison to any other time points (Fig 4A–4F), and at 3 h after rTenascin33 exposure likewise (Fig 4G–4I). Consequently, it could readily be deduced that Tenascin15/30/33 might act as virulence factor related to Giardia-induced IEC apoptosis.
HT-29 and Caco-2 cells were unexposed or exposed to Giardia trophozoites at a multiplicity of infection of 10 and rTenascin15/30/33 at a concentration of 20 μg/mL for 6 h. The exposed cells were examined for early and late apoptotic changes in contrast to unexposed controls (scale bar = 2 μm). “Control” was abbreviated as “Ctr”, “Nucleus” as “Nu”, and “Giardia” as “GI” here.
rTenascin15/30/33 at a concentration of 20 μg/mL was applied. (A, D, G) Exposure of HT-29 and Caco-2 cells to rTenascin15/30/33 promoted activation of PARP and CASP3 as assessed by western blot and gray value analyses. The comparisons were made with the first group of the graph. (B, C, E, F, H, I) Apoptotic effects on HT-29 and Caco-2 cells by rTenascin15/30/33 exposure were assessed by AO/EB (scale bar = 1000 μm) staining and Annexin V-FITC/PI staining. Data from triplicate wells from a representative of three independent experiments are presented as means ± SD. * p < 0.05, ** p < 0.01.
The association of EGFR activation with rTenascin15/30-induced IEC apoptosis
We also explored the potential for recombinant tenascins in EGFR activation. EGFR proteins phosphorylated after exposure of HT-29 and Caco-2 cells to rTenascin15/30/33 and phosphorylation peaked almost simultaneously at 30 min for rTenascin15/30 and at 45 min for rTenascin33 (Fig 5A–5C). Exposure of HT-29 and Caco-2 cells to rTenascin15/30 resulted in an increase in EGFR expression within 9 h and it peaked at 6 h or 9 h depending on cell types (Fig 5A, 5B, 5D and 5E). An increased EGFR expression was found in HT-29 and Caco-2 cells exposed to rTenascin33 within 3 h and it also peaked at 3 h (Fig 5C and 5F). We then explored the links between EGFR activation and HT-29 cell apoptosis during tenascin exposure. EGFR inhibition by Ag1478 was shown to be able to reverse HT-29 cell apoptosis induced by a 6-h exposure to rTenascin15 and rTenascin30 (Fig 6A–6F), while this is not the case for rTenascin33 (Fig 6G–6I). Thus, it is reasonable to infer that rTenascin15/30-induced IEC apoptosis was dependent on EGFR activation, rather than rTenascin33.
Exposure of HT-29 and Caco-2 cells to rTenascin15/30/33 at a concentration of 20 μg/mL was able to promote activation of EGFR as determined by western blot and gray value analyses. The comparisons were made with the first group of the graph. Data from triplicate wells from a representative of three independent experiments are presented as means ± SD. * p < 0.05, ** p < 0.01.
rTenascin15/30/33 was used for exposure at a concentration of 20 μg/mL. (A, D) PARP and CASP3 activation in HT-29 cells induced by a 6-h rTenascin15/30 exposure could be reversed by application of the EGFR inhibitor Ag1478 as assessed by western blot and gray value analyses. The comparisons were made as indicated. (B, C, E, F) HT-29 cell apoptosis induced by a 6-h rTenascin15/30 exposure could be reversed by application of the EGFR inhibitor Ag1478 as determined by AO/EB (scale bar = 1000 μm) and Annexin V-FITC/PI analyses. (G) Activation of PARP and CASP3 in HT-29 cells induced by a 3-h rTenascin33 exposure could not be blocked by application of the EGFR inhibitor Ag1478 as determined by western blot and gray value analyses. (H, I) HT-29 apoptosis induced by a 3-h rTenascin33 exposure could not be blocked by application of the EGFR inhibitor Ag1478 as determined by AO/EB (scale bar = 1000 μm) and Annexin V-FITC/PI analyses. Data from triplicate wells from a representative of three independent experiments are presented as means ± SD. ** p < 0.01. “Control” was abbreviated as “Ctr”, and “not significant” as “ns” here.
rTenascin15/30 exposure promoted EGFR translocation to the nucleus of IECs
It was determined earlier that recombinant tenascins were able to interact with EGFR and involve EGFR activation. Here in immunofluorescence analysis, there existed apparent colocalization of rTenascin15/30/33 with EGFR in HT-29 cells as indicated by red arrows (Fig 7A–7C). It is also of interest to note that a prolonged exposure of HT-29 cells to rTenascin15/30 could lead to EGFR translocation to the nucleus as indicated by yellow arrows, while this is not the case for rTenascin33 (Fig 7D–7F), in agreement with our earlier finding that rTenascin15/30 rather than rTenascin33 was found to be responsible for EGFR-dependent IEC apoptosis.
HT-29 cells were exposed to rTenascin15/30/33 at a concentration of 20 μg/mL. (A, B, C) Colocalization of rTenascin15/30/33 with EGFR was analyzed by immunofluorescent staining (scale bar = 50 μm). (D, E, F) rTenascin15/30 exposure promoted EGFR translocation to the nucleus, while this is not the case for rTenascin33, as determined by immunofluorescent staining (scale bar = 50 μm).
Regulation of EGFR-STAT3 axis in rTenascin15/30-induced IEC apoptosis
Similar to EGFR activation, STAT3 signaling was also confirmed to be activated in HT-29 and Caco-2 cells exposed to rTenascin15/30/33, and the phosphorylation levels of STAT3 varied after tenascin exposure for different time durations (Fig 8A; S5 File, panel A). EGFR inhibition by Ag1478 could significantly repress STAT3 activation evoked by rTenascin15/30 rather than rTenascin33 (Fig 8B; S5 File, panel B). We then explored the relation of STAT3 signaling to HT-29 cell apoptosis induced by recombinant tenascins. STAT3 inhibition by inhibitor Stattic could significantly alleviate HT-29 cell apoptosis induced by rTenascin15/30 rather than rTenascin33 (Fig 8C). Considering our formerly established close association between EGFR activation and rTenascin15/30-induced IEC apoptosis, the collective data suggested an important mechanism of EGFR-STAT3 axis regulation. EGFR nucleus translocation in HT-29 induced by rTenascin15/30 rather than rTenascin33 was observed in western blot analysis (Fig 8D; S5 File, panel C), in line with the observations in immunofluorescence analysis earlier. Likewise, rTenascin15/30 rather than rTenascin33 showed a promotive effect on the induction of translocation of STAT3 to the nucleus (Fig 8D; S5 File, panel C). It is therefore apparent that EGFR/STAT3 nuclear translocation might involve rTenascin15/30-induced IEC apoptosis.
HT-29 and Caco-2 cells were exposed to rTenascin15/30/33 at a concentration of 20 μg/mL. (A) recombinant tenascin exposure promoted STAT3 activation in HT-29 and Caco-2 cells as determined by western blot and gray value analyses. The comparisons were made with the first group of the graph. (B) STAT3 activation in HT-29 cells induced by a 6-h rTenascin15/30 exposure could be reversed by application of the EGFR inhibitor Ag1478, while this is not the case for that induced by a 3-h rTenascin33 exposure, as assessed by western blot and gray value analyses. The comparisons were made as indicated. (C) PARP and CASP3 activation in HT-29 cells induced by a 6-h rTenascin15/30 exposure could be reversed by application of the STAT3 inhibitor Stattic, while this is not the case for that induced by a 3-h rTenascin33 exposure, as assessed by western blot and gray value analyses. The comparisons were made as indicated. (D) rTenascin15/30 exposure promoted EGFR/STAT3 translocation to the nucleus of HT-29 cells, while this is not the case for rTenascin33, as assessed by western blot and gray value analyses. The comparisons were made with the first group of the graph. Data from triplicate wells from a representative of at least three independent experiments are presented as means ± SD. * p < 0.05, ** p < 0.01. “Ctr” read “Control”, “ns” read “not significant”.
Discussion
It is well known that the adhesion of Giardia trophozoites to IECs drives the onset and progression of giardiasis [10–12]. Although Giardia-induced IEC apoptosis and apoptosis-dependent epithelial barrier disruption are believed to play important parts in the pathogenesis of giardiasis [27,30,34,41–50], the causative factors have not yet been entirely elucidated. During Giardia-host interactions, IECs can be driven to undergo apoptosis by ESPs secreted by this extracellular parasite [10–12]. Giardia-secreted cathepsin B-like cysteine protease, giardipain-1, has been identified as a virulence factor influencing tight junction and barrier function of IECs and causing apoptotic damage [41]. Our former studies have used in vitro models of IECs to identify the intracellular signals or modulators that promote or inhibit Giardia-induced apoptosis [46–50]. A recent study confirmed the contributing effects of ESPs, notably pyridoxamine 5’-phosphate oxidase, on the elicitation of giardiasis-related pathogenic responses, including intrinsic apoptosis [45]. Despite those advances in elucidating the molecular underpinnings of giardiasis, the importance of a new class of virulence factors predicted through secretome [13–16], the tenascins with abundant EGF-like repeats, as contributing factors to Giardia pathogenesis, has often been overlooked. The present study demonstrated the association of Giardia-secreted tenascins with apoptotic induction of IECs at both the morphological and molecular levels. rTenascin15/30/33-exposed IECs displayed typical morphological hallmarks of apoptosis, especially cytoplasmic vacuolization and nuclear fragmentation. Strikingly, the following molecular evidence suggested that IEC apoptosis seemed to be initiated at an earlier time point by rTenascin33 compared to rTenascin15/30. The findings here would be an important addition to the complex and intersecting network of giardiasis-causing factors in terms of apoptosis induction.
While EGFR activation or overexpression has been extensively studied for its promotive role in cell proliferation, survival, differentiation, and migration in both normal and malignant contexts [51,52], there were convincing evidences supporting EGFR-dependent apoptosis [53–57]. Here in this study, a significant increase in EGFR expression was seen in IECs exposed to Giardia trophozoites and rTenascin15/30/33, and phosphorylation of EGFR occurred within 1 h. While Co-IP analysis and IFA depicted potential interaction between EGFR and rTenascin15/30/33, further research is needed to fully elucidate the precise underlying interaction mechanism. Interestingly, EGFR activation was then discovered to be implicated exclusively in promoting apoptosis in IECs exposed to trophozoites and rTenascin15/30 rather than rTenascin33, implying the regulatory complexity of EGFR activation on apoptosis induction. The differential roles of rTenascin15/30/33 in inducing EGFR-dependent IEC apoptosis may be directed to the phylogenetic clustering pattern of tenascin families, with a highly divergent clade formed for Tenascin33 [13]. In addition, Tenascin33 has been proposed to possess EGF activity due to a strict correspondence of one of its EGF-like domains with a human-derived biologically active domain CX7–8CX4–8CX10–13CXCX8C [18,22,23], which might contribute to the inactive effect on the induction of EGFR-dependent apoptosis in human IEC model. However, additional efforts should be devoted to the identification of EGFR-independent regulatory pathway by which Tenascin33 would exert an apoptosis-inducing effect on IECs, such as Toll-like receptors (TLRs), because TLR2 activation has been linked to Giardia-induced IEC apoptosis [46]. It is also of interest to note here that exposure of IECs to rTenascin15/30 rather than rTenascin33 could promote the entry of EGFR into the nucleus as confirmed by IFA and western blotting, which might answer for the EGFR-dependent apoptosis-inducing effect of the former.
In congruency with EGFR, activated STAT3 signaling has positive and negative effects on cell proliferation and survival as well [58–61]. Here, like EGFR, STAT3 activation was also determined to be involved in exacerbating apoptosis in IECs exposed to rTenascin15/30 rather than rTenascin33. Translocation of STAT3 into the nucleus was observed in IECs exposed to rTenascin15/30 rather than rTenascin33 as well. Increased ratio of Bax to Bcl-2 and enhanced cleavage of the downstream apoptotic initiator CASP9 and effector CASP3 involve activation of Giardia-induced intrinsic IEC apoptosis as described before [49]. Whether such apoptotic process results from the positive regulatory function of STAT3 in Bax expression needs future exploration. Whatever the case, it has been evident that Fusobacterium nucleatum-aggravated experimental colitis occurs as a consequence of infection-triggered STAT3 activation and nuclear translocation [62]. This study further confirmed EGFR as a positive regulator of STAT3 activation during exposure of IECs to recombinant tenascins. Taken these findings together, it is not difficult to speculate that EGFR and STAT3 might form a signaling axis to positively regulate Giardia-evoked IEC apoptosis. In reality, it has been previously noted that EGFR and STAT3 can function together in apoptotic regulation [35,63]. TLR4-COX-2/HSP70 axis-mediated anti-apoptotic function has been proposed by our former studies as a defense strategy against Giardia infection [46,47], which might counteract the EGFR-STAT3 axis-mediated apoptosis-inducing impact, thereby facilitating disease tolerance and preserving host homeostasis. Intriguingly, beyond apoptosis-inducing function, activation of EGFR-STAT3 axis in Toxoplasma gondii-infected cells can prevent the parasite from being targeted by autophagic mechanisms [64].
In conclusion, herein, it was initially observed that EGFR activation was involved in Giardia-induced IEC apoptosis. We then examined the interaction of tenascins with EGFR and elucidated the vital role of recombinant Giardia tenascins in evoking IEC apoptosis. EGFR activation and nuclear translocation were demonstrated to be associated with rTenascin15/30-induced IEC apoptosis, rather than rTenascin33. STAT3 activation and nuclear translocation were then proven to function together with EGFR in promoting rTenascin15/30-induced IEC apoptosis, rather than rTenascin33. This study uncovered the differential roles of Giardia-secreted tenascins in intensifying EGFR-STAT3 axis-dependent IEC apoptosis, expanding current knowledge about the pathogenesis of giardiasis and deepening our understanding of the underlying mechanisms of interactions between noninvasive Giardia and IECs. Of physiological relevance, Giardia tenascins-related apoptosis induction would accelerate the host defense-related renewal of IECs. Notwithstanding the importance of the findings, further assessment based on organoid-derived monolayers or an accessible laboratory animal model susceptible to infection with G. duodenalis WB isolate is required to confirm the observations.
Supporting information
S1 File. The original uncropped images of western blots corresponding to Fig 1A/1D/1E/1F, Fig 4A/4D/4G, Fig 5A/5B/5C/5D/5E/5F, Fig 6A/6D/6G, and Fig 8A/8B/8C/8D.
https://doi.org/10.1371/journal.pntd.0014153.s001
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S2 File. Cell viability analysis for the effect of inhibitor treatment.
(A) Cell viability of HT-29 was assessed by CCK-8 assay when different concentrations of Ag1478 were used. (B) Cell viability of HT-29 was assessed by CCK-8 assay when different concentrations of Stattic were used.
https://doi.org/10.1371/journal.pntd.0014153.s002
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S3 File. SDS-PAGE analysis of expression and purification of rTenascin15/30/33 and endotoxin testing prior to recombinant protein challenge.
(A) M, protein marker; Lane 1, total protein fractions after IPTG induction; Lane 2, pooled fractions from the nickel column; Lane 3, the wash and elution fractions; Lanes 4–7, purified recombinant His6-tagged proteins with their molecular weights shown beside the arrows. (B) M, protein marker; Lanes 1–3, the purified and concentrated recombinant proteins targeting Tanascin15, Tanascin30, and Tanascin33, respectively, with their molecular weights shown beside the arrows. (C) The recombinant tenascins used for exposure were tested to be free of endotoxins that would interfere with experimental observation.
https://doi.org/10.1371/journal.pntd.0014153.s003
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S4 File. The interaction of rTenascin15/30/33 with EGFR in HT-29 cells was assessed by Co-IP.
https://doi.org/10.1371/journal.pntd.0014153.s004
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S5 File. Supplementary data supporting the findings of Fig 8.
(A and B) Temporal analyses of changes in the ratios of p-STAT3 to STAT3 referred to Fig 8A and Fig 8B. * p < 0.05, ** p < 0.01. “Ctr” read “Control”, “ns” read “not significant”. (C) Assessment of the nucleus-cytoplasm separation quality by western blotting referred to Fig 8D.
https://doi.org/10.1371/journal.pntd.0014153.s005
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Acknowledgments
Wei Li acknowledges the Longjiang Scholar (Distinguished Professorship) Award obtained in 2025 and the contributions of all other individuals and organizations who assisted in the research process.
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